chapter eight: microbial genetics

1. chapter eight: microbial genetics

2. the hereditary materialGriffith 1927 & Avery, et al. 1944 the “transforming principle” coined by Griffith, identified by Avery

3. the hereditary materialHershey Chase, 1952

4. the bacterial chromosome

5. plasmids • F factor (conjugative plasmid) • dissimilation plasmids • R factors

6. horizontal & vertical gene transfer

7. antiparallel replication

8. vertical gene transfer (VGT): DNA replication synthesis requires primers & the 3΄ OH

9. horizontal gene transfer (HGT): gene expressionsimultaneous transcription & translation

10. HGT: recombination

11. RecA & chromosomal recombination

12. insertion sequences & jumping genes

13. recombination: transformation

14. recombination: transduction

15. recombination: conjugation

16. genetic transfer

17. regulating bacterial gene expression: constitutive enzymes  operons

18. regulating gene expression* * decreased levels of cellular glucose create high cAMP levels which further regulate the expression of lactose catabolizing enzymes- this will not be discussed in this class

19. transcriptional control inducible operon: effector effects by inhibiting repressor = inducer repressible operon: effector effects by activating repressor = corepressor

20. quorum sensing & gene regulation • B. subtilis sporulation •  cell density =CSF &  ComX  ComS  competence •  cell density &  CSF = ComS inhibited  sporulation • Gram negative biofilm formation •  acylated homoserine lactones(HSLs) in loss of flagella • sessile microbes initiate biofilmformation • P. aeruginosa virulence • high cell density activates virulence genes  disease

21. Chapter Eight Learning Objectives • What did the work of Griffith, Avery and Hershey & Chase contribute to the field of biology? • How is the bacterial chromosome different from the eukaryotic chromosome? What other molecule contains useful genetic information for prokaryotes? Compare and contrast DNA replication in eukaryotes vs. prokaryotes. • Why does the replication of every DNA molecule start with a short segment of RNA? • Define: vertical gene transfer, horizontal gene transfer, DNA replication, gene expression, transcription, translation, conjugation, transduction and transformation. • How is gene expression in prokaryotes different from eukaryotes, both in the timing of transcription & translation and in how transcription is regulated? • How do the RecA protein and transposons enable novel DNA to be integrated and used in the recipient cell? Discuss this for both transformation and transduction. • Define F factor, F+ cell, F- cell and Hfr cell. Understand what happens when F+ , F- & Hfr cells interact during conjugation. • Describe the mechanisms of inducible and repressible operons. Include the role of promoters, operators, effectors, inducers, repressors and co-repressors in your answer. • Discuss the levels of bacterial control of gene expression, paying particular attention to post-translational and transcriptional control, as discussed in lecture. • What is quorum sensing? How does it relate to gene expression, particularly as relates to sporulation, biofilm formation, competence and virulence genes.

22. chapter nine: biotechnology

23. biotechnology and recombinant DNA • biotechnology: using recombinant DNA (rDNA) cells • using vectors to produce clones • therapeutic applications • human enzymes and other proteins • subunit vaccines • viral DNA vaccines • gene therapy • disease ID • mutant screening • natural or mutagen-induced • cloning & expression  recombinant DNA technology

24. rDNA technology

25. pharmaceutical products

26. making rDNA: restriction endonucleases in vivo: defense system, cut only non-methylated DNA in vitro: molecular scissors

27. moving rDNA: plasmid vectors

28. shuttle vectors

29. finding rDNA: blue/white colony selection pBluescript™ vectors

30. moving rDNA: viral vectors

31. pathogen detection: PCR

32. RNA pathogen detection reverse transcriptase RNA genome DNA template

33. Norovirus outbreak RT-PCR with Norovirus primer

34. Chapter Nine Learning Objectives • Define biotechnology & recombinant DNA technology. What applications were discussed in lecture which utilize this technology? • Discuss how recombinant DNA molecules are made using restriction enzymes. What are the steps used in making these recombinant molecules? How do both plasmids & viruses play a role in expressing recombinant DNA molecules? • There are three essential regions on a shuttle vector. What are they, and what do they do? How do they help to identify in vitro transformed cells? • Describe the process of PCR and the use of reverse transcriptase to amplify an RNA template. How can these technologies be used to identify a microbial pathogen?

35. chapter eight: mutagens & mutation screening

36. mutation frequency • change in the genetic material • spontaneous • no mutagen • 109 per bp • 106 genes • mutagens  freq.105 – 103 per gene

37. mutation types • base substitution (point mutation) • silent • 3rd G to any other base = glycine (redundancy) • protein change • missense, nonsense, frameshift mutation

38. mutagens

39. mutation repair • photolyase repair • separate thymine dimers • nucleotide excision repair • various damage repaired • UvrA, UvrB, UvrC, UVrD(DNA helicase) • SOS recA repair • cell cycle arrested • DNA repair & mutagenesis induced

40. replica plating: negative mutant selection negative (indirect) selection: mutants do not grow

41. the Ames test: positive mutant selection & carcinogen identification positive (direct) selection: mutants grow or appear different

42. Chapter Eight Learning Objectives • Define: silent, missense, nonsense and frameshift mutation. How can these errors be repaired in a cell? • How does the term auxotroph relate to mutant selection? • Why is replica plating necessary for the indirect selection of mutants? • What is the Ames test? How and why does it result in positive mutant selection?